Capillary electrophoresis is a well-known procedure for separation of chemical components. A sample solution containing molecules to be separated is placed in a length of capillary tubing containing an electrophoretic medium. Upon application of an electric field across the capillary, different components within the sample migrate at distinct rates towards the oppositely charged end of the capillary dependent upon their relative electrophoretic mobilities in the electrophoretic medium. Due to the varying electromigratory rates, the sample components become increasingly separated into distinct zones or groups as they progress along the capillary. At some position along the capillary, the components of the sample are detected.
Electrophoresis has been applied to the separation of charged materials such as proteins, nucleic acids, and cells. These separations depend upon differences in charge density, molecular size, and partitioning or complexation with a mobile phase additive. U.S. Pat. No. 5,061,361 relates to a capillary zone electrophoresis system in which a nanoliter volume of sample is introduced into the capillary tube, and an electric field is imposed on the system to effect separation of the charged components. After migration along the length of the tube, the sample components are detected via ultra-violet absorbance. U.S. Pat. No. 5,084,150 relates to an electrokinetic method of separation in which the surface of moving charged colloidal particles is treated so as to interact selectively with the sample molecules to be separated. An electric field is imposed on a capillary tube containing the colloidal particles and the sample to achieve separation. U.S. Pat. No. 5,045,172 relates to a capillary electrophoresis apparatus in which electrodes are attached at each end of a capillary tube, and a detector is coupled to the tube. U.S. Pat. No. 4,181,589 relates to a method for separating biological cells using an electric field. The above-described U.S. patents are hereby incorporated by reference.
Various types of assays are used as clinical diagnostics. Immunoassays of various formats widely are used in clinical diagnosis to measure analytes in body fluids. Immunoassays also are used for the sensitive and specific measurement of analytes in complex mixtures in research, industrial and environmental applications. For example, an enzyme linked immunosorbent assay ("ELISA") has been used to determine levels of cytokines, Thyroid-Stimulating Hormone ("TSH") and other analytes in serum and other biological samples. Engvall, Methods in Enzymology, 70:419 (1980); Schurrs and VanWeeman, J. Immunoassay, 1:229 (1980); and Scharpe, et al., Clin. Chem., 22:733 (1976) are general references to enzyme immunoassay techniques and are herein incorporated by reference in their entirety.
Patent Cooperation Treaty ("PCT") publications WO 93/220553 and WO 93/22054, and U.S. Pat. No. 5,304,487, herein incorporated by reference, describe various types of assays performed in microscale analytical devices. Devices for detecting the presence of a preselected analyte in a fluid sample or for analyzing a fluid cell containing a sample are disclosed. The devices typically are made of a solid substrate that is microfabricated to have a sample inlet port and a mesoscale flow system. Other embodiments have various flow restriction designs. The solid substrate may be a few millimeters thick and about 0.2 to 2.0 cm square. The flow channel typically on the order of 0.1 .mu.m to 500 .mu.m. The devices are used in a wide range of automated, sensitive and rapid tests using various flow inducing means.
Immunoassays using electrophoretic separation of free and bound antigen have been explored. Assays using capillary electrophoresis have provided rapid separation and accurate quantitation. Moreover, the microvolume scale of capillary electrophoresis reduces the amount of sample and reagents required, as well as decreasing the amount of waste generated. However, many assays require analyte quantitation to be at levels of 10.sup.-10 to 10.sup.-12 Molar. Given these low concentrations of analyte, even laser induced fluorescence detection coupled with capillary electrophoresis may not be sufficient for the detection of an analyte. Overall, sensitivity, speed and cost are important factors to be considered in designing and conducting an immunoassay.